GTP-binding proteins are key switches in signaling pathways that regulate critical cellular functions such as growth and differentiation. Ras, which serves as a prototype for GTP-binding is constitutively activated in a large number of cancers including those of the pancreas, bladder, colon, and lung. The long-term goal of this research is to interfere with the oncogenic forms of Ras in human disease by understanding its cycling. Because oncogenic Ras relies on its intrinsic ability to hydrolyze and exchange GTP, the primary goal of this research proposal is to better our understanding of Ras cycling between the active and inactive states by revealing structures of intermediates of the reactions of GTP hydrolysis and guanine nucleotide exchange. Screening of drugs that will stabilize a non-signaling conformation of oncogenic Ras will be also performed. This proposal is based on findings that altering the flexibility of a hinge region stabilizes structures of Ras that are normally transient. Using this approach, two structures of intermediates along the path for GTP hydrolysis were stabilized as well as an open non-signaling conformation, which is also adopted by the native protein. The first aim of this proposal tests the hypothesis that the open conformation of Ras mimics the structure of an intermediate for nucleotide exchange and the structu-e of an unappreciated native conformation. Additional structures of intermediates for Ras cycling will be generated by altering the flexibility of another hinge region. The generated mutants will be studied using a combination of structural, molecular dynamics, biochemical, and in vivo techniques. The second aim tests the hypothesis that Rho- family members and trimeric G-proteins do not follow the Ras path of GTP hydrolysis and nucleotide exchange despite strong sequence homology. The third aim uses biophysical, structural, and cellular approaches to study how small molecules identified by screening the NCI database interact with the open non-signaling conformation and inhibit oncogenic Ras in a pancreatic cancer model system. The outcome of the proposed research should improve our understanding of the regulation of key signaling proteins and our ability to interfere with their action in human diseases.